**2. Materials and methods**

**Figure 2** illustrates the experimental design. Briefly, this study was carried out using sparkling wines from nine consecutive vintages (2008–2016) from the Juve & Camps winery (AOC Cava, Sant Sadurní d'Anoia, Barcelona, Spain). All these

**Figure 2.** *Experimental design.*

sparkling wines were produced with grapes from the same vineyards and were elaborated as similarly as possible. The youngest sparkling wine (2016) was disgorged 3 months after "tirage" and the sparkling wines from the other vintages were also disgorged 3 months after having completed 1–8 years of aging, respectively. In all the cases the lees were recovered, washed, resuspended in a model wine solution and bottled for subsequent analysis. These bottles were inserted with a pill for measuring dissolved oxygen by luminescence (Nomasense TM O2 Trace Oxygen Analyzer). Aliquots of the lees from the nine consecutive vintages were also used for ultrastructural observation using scanning electron microscopy [15]. Consequently, this study was performed with sparkling wines and lees from the first to the ninth years of aging.

The sparkling wines were used for color [26], polysaccharides [27] and proteins [28] analysis, for measuring the foaming properties [29] and for tasting. In parallel, the oxygen concentration was measured periodically in the bottles in which the lees were transferred [22]. Exactly 1 year later the solution was centrifuged and used for polysaccharides and protein analysis [27, 28].
